PROJECT SUMMARY Coronaviruses (CoVs) are a family of positive-sense RNA viruses that cause human illnesses ranging from the common cold to severe and lethal respiratory disease. Since 2002, two CoVs (SARS- and MERS-CoV) have emerged as zoonoses with pandemic potential, and SARS-like CoVs continue to circulate in bats. Lack of proofreading activity greatly increases the genetic diversity of most families of positive-strand RNA viruses and is thought to limit genome size, facilitate adaptability and virulence, and alter host range. Coronaviruses, whose genomes are several times larger than other positive-strand RNA viruses, encode a unique proofreading exoribonuclease (ExoN) in nonstructural protein 14 (nsp14-ExoN) that is distinct from the nsp12- RNA-dependent RNA polymerase (nsp12-RdRp). We have demonstrated that inactivation of ExoN [ExoN(-)] results in a mutator phenotype characterized by increased mutation frequency, increased sensitivity to RNA mutagens, decreased replication, decreased fitness, and stable attenuation in vivo. The overall goal of this proposal is to understand how nsp14-ExoN and nsp12-RdRp facilitate replication, fidelity, and overall fitness of coronaviruses. We have generated a murine hepatitis virus lacking ExoN [MHV-ExoN(-)] and passaged the virus 250 times (P250), resulting in a mutant MHV with compensated replication and restored resistance to mutagens. MHV-ExoN(-)-P250 retains the engineered ExoN(-) mutations and encodes additional mutations in nsp14 and nsp12-RdRp. In Specific Aim 1, we will define determinants within nsp12-RdRp and nsp14-ExoN that compensate for ExoN-associated defects in replication, RNA synthesis, and replication fidelity using WT- MHV, MHV-ExoN(-), MHV-ExoN(-)-P250, as well as engineered MHV that exchanges specific P250 proteins in the isogenic MHV-ExoN(-) background. In Specific Aim 2, we will determine how ExoN proofreading affects the genetic diversity and overall fitness of coronavirus populations. We will use highly accurate deep sequencing to examine the types and abundance of mutations in our altered-fidelity viruses. We will also quantitatively measure replication in hundreds of single-cell infections to determine how genetic diversity impacts the success and extent of individual infection events. Together, these studies will define determinants within the RdRp and nsp14-ExoN that cooperate to regulate RNA synthesis and replication fidelity. Further, these results will define the role of ExoN in regulating CoV genetic diversity and elucidate how genetic diversity affects virus fitness. Finally, these studies will establish new tools for the incisive study of CoV replication and fitness, and will define new vulnerabilities in CoVs that can be targeted by viral inhibitors or exploited for vaccine development.